The ATP-Waiting Conformation of Rotating F1-ATPase Revealed by Single-Pair Fluorescence Resonance Energy Transfer

F1-ATPase is an ATP-driven rotary motor in which a rod-shaped γ subunit rotates inside a cylinder made of α3β3subunits. To elucidate the conformations of rotating F1, we measured fluorescence resonance energy transfer (FRET) between a donor on one of the three βs and an acceptor on γ in single F1molecules. The yield of FRET changed stepwise at low ATP concentrations, reflecting the stepwise rotation of γ. In the ATP-waiting state, the FRET yields indicated a γ position ≈ 40 ° counterclockwise (= direction of rotation) from that in the crystal structures of mitochondrial F1, suggesting that the crystal structures mimic a metastable state before product release.